Pleiotropic, non‑cell death‑associated effects of inhibitors of receptor‑interacting protein kinase 1 in the heart
C. Horvath1 · A. Szobi1 · L. Kindernay2 · T. Ravingerova2 · A. Adameova1,2

Received: 24 November 2020 / Accepted: 11 March 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Inhibition of receptor-interacting protein kinase 1 (RIP1) has been recognized as a compelling tool for limiting necroptosis. Recent findings have indicated that RIP1 inhibitor, necrostatin-1 (Nec-1), is also able to modify heart function under non- cell death conditions. In this study, we investigated its underlying molecular mechanisms and compared with those of novel pharmacologically improved agents (Nec-1s and GSK’772) and its inactive analog (Nec-1i). Heart function was examined in Langendorff-perfused rat hearts. Certain proteins regulating myocardial contraction–relaxation cycle and oxidative stress (OS) were evaluated by immunoblotting and as the extent of lipid peroxidation, protein carbonylation and nitration, respectively. In spite of the increase of left ventricular developed pressure (LVDP) due to treatment by both Nec-1 and Nec-1i, only the former agent increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) at threonine 287 and cardiac myosin-binding protein-C (cMyBPc) at serine 282. In contrast, Nec-1s did not elicit such changes, while it also increased LVDP. GSK’772 activated CaMKIIδ-phospholamban (PLN) axis. Neither protein kinase A (PKA) nor its selected molecular targets, such as serine 16 phosphorylated PLN and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), were affected by either RIP1 inhibitor. Nec-1, like other necrostatins (Nec-1i, Nec-1s), but not GSK’772, elevated protein tyrosine nitration without affecting other markers of OS. In conclusion, this study indicated for the first time that Nec-1 may affect basal heart function by the modulation of OS and activation of some proteins of contraction–relaxation cycle.
Keywords RIP1 · Necrostatins · GSK’772 · Oxidative stress · Contraction–relaxation cycle · Heart

Abbreviations
BP Blood pressure
CaMKIIδ Ca2+/calmodulin-dependent protein kinase II delta
cMYBPc Cardiac myosin-binding protein-C DNPH 2,4-Dinitrophenylhydrazine
ECC Excitation–contraction coupling EG Electrogram
GSK’772 GSK2982772
HR Heart rate
IDO Indoleamine 2,3-dioxygenase
LVDP Left ventricular developed pressure

 A. Adameova [email protected]
1 Department of Pharmacology and Toxicology, Faculty
of Pharmacy, Comenius University in Bratislava, Odbojarov 10, 832 32 Bratislava, Slovak Republic
2 Centre of Experimental Medicine, Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovak Republic

Nec-1 Necrostatin-1
Nec-1i Necrostatin-1i
Nec-1s Necrostatin-1s
OS Oxidative stress
PKA Protein kinase A
PLN Phospholamban
PVDF Polyvinylidene difluoride
RIP1 Receptor-interacting protein kinase 1 TBARS Thiobarbituric acid reactive substances SERCA2a Sarco/endoplasmic reticulum Ca2+-ATPase

Introduction
Receptor-interacting protein kinase 1 (RIP1) is a multi- functional serine/threonine kinase that has emerged as a key mediator of cell death due to necroptosis [1], apoptosis [2], and inflammatory response [3]. In addition, it has been suggested to function also as a pro-survival molecule in a nuclear factor kappa B-independent [4] and -depend- ent manner [5] by regulating gene transcription program

and protein synthesis processes culminating in blockade of cell death and promotion of other cytoprotective mecha- nisms. Post-translational modifications of RIP1 determine the predominance of one or more of these intracellular events. Ubiquitination of the signaling adaptor RIP1 pro- motes cytoprotective signaling, while disruption of RIP1 ubiquitination as well as phosphorylation on Ser161 and caspase-8-mediated cleavage of the kinase domain con- verts this protein kinase into a death-inducing molecule [6, 7].
The inhibition of phosphorylation of RIP1 on Ser161 by
necrostatins has been reported to be an effective tool for prevention/retardation of necroptotic cell death [6, 8–10]. Necrostatin-1 (Nec-1), the first-synthetized and most widely used RIP1 inhibitor, exhibits remarkable cardio- protective effects due to such anti-necroptotic action [9, 11–13]. Another necrostatin, Nec-1i, serving as a suit- able control substance to Nec-1, lacks the RIP1 inhibitory effect due to removal of a single methyl moiety [8]. Both Nec-1 and Nec-1i have been shown to display additional RIP1-independent effects by suppressing the activity of the immunomodulatory enzyme indoleamine 2,3-dioxy- genase (IDO) [14], thereby potentially affecting other cel- lular events. In contrast to classic RIP1 inhibitors, novel agents, such as Nec-1s, a more metabolically stable and potent necrostatin compared to other necrostatin analogs, as well as GSK2982772 (GSK’772), a structurally differ- ent RIP1 inhibitor currently being in three phase2a clini- cal trials (NCT02858492; NCT02776033; NCT02903966),
lack such off-target IDO-modulating action [8, 15]. This feature might make these two innovative RIP1 inhibitors pharmacologically superior to Nec-1 and Nec-1i.
Previous studies have examined non-cell death-related effects of Nec-1 and Nec-1i, showing that the administra- tion of the former, but not the latter substance, is capa- ble of temporarily increasing both systolic and diastolic blood pressure (BP) in anesthetized rats [11, 16]. Like- wise, Nec-1 has modified some ECG parameters suggest- ing that it acts on cardiac pacemaker cells [16]. Based on these findings we hypothesize that Nec-1 might have some pleiotropic effects directly affecting heart function. Therefore, a molecular analysis of certain Ca2+ cycling proteins, together with the assessment of oxidative stress (OS), which plays a pivotal role in both cardiovascular physiology and pathology [17–19], has been performed. In addition, novel RIP1 inhibitors given at equipotent doses were also tested to investigate whether there is a drug class-related mechanism of action. These findings can reveal additional intracellular action of RIP1 and provide valuable information on pharmacodynamic action of the investigated RIP1 inhibitors what can be important from the point of view of safety of these drugs.

Methods
Chemicals and reagents

Drugs used were sourced from Selleck Chemicals (GSK2982772, Nec-1s; USA) and Merck (Nec-1, Nec-1i; DE). Unless otherwise specified, all other chemicals used were supplied by CentralChem (SR), Merck (DE), Apollo Scientific (UK), Sigma-Aldrich (DE), or Alfa Aesar (USA) in the highest available purity.

Experimental groups

Male Wistar rats weighing 275 ± 25 g (14–16 weeks old; Dobrá Voda, SR) were housed under standard conditions (22 ± 2 °C temperature, 55 ± 5% humidity) with a daily 12-h light/dark cycle. Animals were fed a standard pelleted diet, had ad libitum water access, and their health was moni- tored daily. After 7 days of adaptation, the rats (33 in total) were randomized into five groups: (a) a vehicle (0.004% v/v DMSO)-treated group (Control, n = 6), (b) a group treated with 1.2 µM of necrostatin-1 (Nec-1, n = 8), (c) a group treated with 1.2 µM of necrostatin-1i (Nec-1i, n = 6),
(d) a group treated with 0.5 µM of necrostatin-1s (Nec-1s, n = 7), and (e) a group treated with 2.5 µM of GSK2982772 (GSK’772, n = 6). The chosen concentration of Nec-1s corresponds to the concentration used in our previously published study [13], while concentrations of Nec-1 and GSK’772, reflecting the murine RIP1 IC50 values of the individual compounds [15, 20], were adjusted to be roughly equipotent with respect to RIP1 inhibition by Nec-1s. The used concentration of Nec-1i, which does not inhibit RIP1, was equal to that of Nec-1, its closest structural analog.

Perfusion protocol

Anesthesia was induced with an i.p. injection of sodium pentobarbital in a dose of 60 mg/kg in combination with heparin. The hearts were then rapidly excised, placed in ice-cold perfusion buffer, cannulated via the aorta and perfused in the Langendorff mode at a constant perfusion pressure of 73 mmHg and temperature of 37.5 ± 0.2 °C. A modified Krebs–Henseleit buffer (pH 7.4; 3.2 mM KCl; 118 mM NaCl; 1.2 mM MgSO4; 1.25 mM CaCl2; 1.18 mM
KH2PO4, 11 mM glucose, 25 mM NaHCO3) saturated with a mixture of 95% oxygen and 5% carbon dioxide was used as the perfusion medium. An initial 20 min stabilization period was followed by a 40-min perfusion with perfusion buffers containing tested compounds as described in the experimental groups section (Fig. 1). As necrostatins are known to be light sensitive, the whole perfusion apparatus

Control Nec-1 Nec-1i
Nec-1s GSK’772
0

Langendorff perfusion

DMSO (0.004% v/v)

Nec-1 (1.2 μM)

Nec-1i (1.2 μM)

Nec-1s (0.5 μM)

GSK‘772 (2.5 μM)

20 60
Time (min)

1.5% w/v SDS; 7.5% v/v glycerol) containing protease (2 mmol/L AEBSF; 5 μg/mL leupeptin; 2 μg/mL pepstatin A) and phosphatase (60 mmol/L NaF; 4 mmol/L Na3VO4) inhibitors. Raw homogenates were centrifuged at 13,000 RCF for 10 min at 4 °C. Lowry assay was used to deter- mine the protein concentration of prepared homogenates. Prepared samples (35 μg/lane) were electrophoresed using SDS-PAGE under reducing conditions (Bis-tris/MOPS gels, concentrations from 8 to 10%) and transferred to PVDF (polyvinylidene difluoride) membranes (Immobilon-P®, Millipore, USA). Membranes were blocked with 2% PVP in TBST for 30 min at room temperature and were incubated with primary antibodies against target epitopes followed by HRP-conjugated secondary antibodies. Chemiluminescent signal detection was performed using a commercial kit (Luminata, Millipore, USA) and myECL Imager (Thermo Fischer Scientific, USA) and analyzed with myImage Analy- sis software (Thermo Fischer Scientific, USA). As loading

Hemodynamic analysis ImmunobloGng
Oxidative stress assessment

Fig. 1 An illustration showing experimental design

and solutions were protected from direct light throughout preparation and use. Left ventricular (LV) pressure was measured by means of a non-elastic water-filled balloon inserted into the left ventricle via the left atrium and con- nected to a pressure transducer (MLP844, ADInstruments, Germany). An epicardial electrogram (EG) was registered by means of two stainless steel electrodes attached to the apex of the heart and the aortic cannula. Heart rate (HR) was calculated from the EG. HR, LV developed pressure (LVDP, systolic minus diastolic pressure), and maximal rates of pressure development [+(dP/dt)max] and fall [−(dP/dt)max] as the indexes of contraction and relaxation were measured with PowerLab/8SP Chart 8 software and used to assess cardiac function. After perfusion, samples of LV tissue were excised, immediately frozen in liquid nitrogen, and stored at − 80 °C until further processing.
SDS‑PAGE and immunoblotting

For semiquantitative protein expression analysis, stored left ventricular samples were pulverized in liquid nitrogen and lysed with a modified RIPA buffer (50 mmol/L MOPS, pH 7.4; 60 mmol/L NaCl; 0.5 mmol/L EDTA; 1 mmol/L EGTA; 1% v/v; Triton X‐100; 0.5% w/v Na deoxycholate;

control, whole-lane post-transfer protein staining intensity (with Ponceau S) was used [21]. Final relative intensities corresponding to protein expression were calculated as a ratio of a target epitope signal to the total protein staining in its lane. The following primary antibodies were used: rabbit anti-pPKA substrate (#9624, Cell Signaling, USA), rabbit anti-CaMKIIδ (15443-1-AP, ProteinTech, USA), rab- bit anti-pThr287CaMKIIδ (PPS002, R&D Systems, USA), mouse anti-cMyBPc (sc-137237, Santa Cruz, USA), rabbit anti-pSer282cMyBPc (ALX-215-057, Enzo Life Sciences, USA), mouse anti-PLN (A010-14, Badrilla, UK), rabbit anti-pSer16PLN (ab92697, Abcam, UK), rabbit anti-pThr- 17PLN (SAB1306553, Sigma-Aldrich, DE), rabbit anti-SER- CA2a (#9580, Cell Signaling, USA), and mouse anti-DNP (MAB2223, Merck, DE), mouse anti-NO2Tyr (MAB5404, Merck, DE). These were used in conjunction with appro- priate secondary antibodies: donkey anti-rabbit[H + L] IgG-HRP (711-035-152, Jackson Immunoresearch, UK) and goat anti-mouse[L] IgG-HRP (115-035-174, Jackson Immunoresearch, UK). Final protein expression results are presented as relative expression changes normalized to the control group mean.
Determination of protein carbonylation and nitration

Protein carbonylation analysis was performed according to Conrad et al. [22]. Briefly, 6 µg of total protein per sample was electrophoresed and transferred to PVDF membranes as during regular Western blotting. Afterward, the mem- branes were washed with water and methanol and left to dry. Dry membranes were then subjected to the derivatization

of carbonyl groups. The membranes were washed in 100% methanol, 20% methanol, and then 6% HCl solution for 5 min each. Subsequently, the membranes were incubated for exactly 5 min in the dark in a derivatization solution (100 μg/mL 2,4-dinitrophenylhydrazine—DNPH, 6% HCl). After derivatization, the membranes were rinsed multiple times with 6% HCl and methanol to remove unreacted DNPH. Derivatized membranes were then used to detect DNP adducts with a specific anti-DNP antibody as is described in Western blotting. Protein nitration was detected directly (24 µg protein/lane) with no extra processing steps by probing regularly prepared PVDF membranes with a spe- cific anti-NO2Tyr antibody. Whole-lane signal positivity was then used to calculate the relative expression of modified protein residues vs. the control group.
Determination of lipid peroxidation by the TBARS method

The level of thiobarbituric acid reactive substances (TBARS) in left ventricular tissue lysates, a marker of lipid peroxidation, was determined by the method of Shlafer and Shepard [23] with minor modifications. First, 40 µL of tissue homogenate (as prepared for Western blotting) was mixed with 40 µL of 20% trichloroacetic acid. Then, 320 µL of TBA reagent (37 mM thiobarbituric acid, 500 mM NaOH, 15% acetic acid) was added and heated at 100 °C for 70 min. After heating, the mixture was allowed to cool down, and the pink, highly specific MDA-(TBA)2 (malon- dialdehyde-(TBA)2) adduct was extracted into 400 µL of a 1-butanol:pyridine (15:1) mixture. Finally, the absorbance of the 1-butanol layer was measured using a spectrophotom- eter (ELx800, BioTek, USA) at 535 nm. All measurements were performed in duplicate, and their average was used to calculate the concentration of MDA equivalents using a calibration curve prepared from the tetrabutylammonium salt of MDA. The final concentration of MDA in the samples was normalized to their protein concentration as determined by the Lowry method. Final results are depicted as relative changes in MDA equivalents relative to the control group mean.
Statistical analysis

Data and statistical analysis comply with the recommenda- tions on experimental design and analysis in pharmacology [24]. Data are expressed as mean ± SEM with the number
(n) of independent biological samples per experiment. One- way ANOVA was used to compare differences among the groups, and when F achieved P < 0.05, Tukey’s post hoc test was used to compare differences between individual pairs of groups. Differences were considered significant when P < 0.05.

Results
Hemodynamic parameters of the isolated rat hearts

Basic hemodynamic parameters of the isolated rat hearts are shown in Table 1. There were no differences in these param- eters among the groups at baseline, prior to the treatment with RIP1 inhibitors. None of the tested agents affected HR of the hearts whose values did not differ from those in the control group, and at 40th min of perfusion they were similar with the baseline values. On the other hand, the increased LVDP values were recorded at this time point in the hearts perfused with necrostatins. In contrast, GSK’772 unlikely modified contractile function of the heart while elicited posi- tive lusitropy as indicated by the significantly higher values of −(dP/dt)max.
Levels of some proteins affecting contraction– relaxation cycle

Incubation with anti-phospho-protein kinase A (PKA) sub- strate showed that the phosphorylation of proteins medi- ated by the cyclic adenosine monophosphate-dependent PKA, which plays a multifaceted role in the regulation of heart function [25], was comparable among all the groups (Fig. 2a, b, h). The target of this protein kinase, phospholam- ban (PLN) phosphorylated at Ser16, was also unchanged regardless of RIP1 being active or inhibited (Fig. 2c, i). Sim- ilar pattern of results was observed in the protein levels of sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) (Fig. 2a, d, j), whose Ca2+ pumping activity is promoted by phosphorylation of PLN resulting in lusitropy [26].
Phosphorylation of Ca2+/calmodulin-dependent protein
kinase II delta (CaMKIIδ), another protein kinase hav- ing a key role in the regulation of excitation–contraction coupling (ECC) of the heart [27] and also being linked to some necroptotic regulators, such as RIP1 and RIP3 [28, 29], was analyzed as a possible target of RIP1 inhibitors. Compared to vehicle- and Nec-1i-treated hearts, the applica- tion of Nec-1 caused an increase in the pThr286-CaMKIIδ/ CaMKIIδ ratio with data being close to statistical signifi- cance (P = 0.0589) (Fig. 2a, e). A significant elevation in pThr286-CaMKIIδ/CaMKIIδ ratio was also observed in the GSK’772 group compared to both Nec-1s- and vehicle- treated hearts (Fig. 2a, k). Even though both Nec-1 and GSK’772 elevated pThr286-CaMKIIδ phosphorylation, the former substance decreased and the latter inhibitor increased the pThr17-PLN/PLN ratio in comparison to vehicle treat- ment (Fig. 2a, f, l). Another target of CaMKIIδ, phospho- cardiac myosin-binding protein-C (pSer282-cMyBPc), which has also been shown to regulate myocardial contraction [30], was significantly increased by Nec-1, while other RIP1

Table 1 Hemodynamic parameters of isolated rat hearts
Baseline values End of perfusion (40 min)

HR [beats/ min]

LVDP
[mmHg]

+(dP/dt)max [mmHg/s]

−(dP/dt)max [mmHg/s]

HR [beats/ min]

LVDP
[mmHg]

+(dP/dt)max [mmHg/s]

−(dP/dt)max [mmHg/s]

Control 262 ± 14 76.7 ± 9.4 1789.0 ± 297.7 1220.2 ± 225.1 256 ± 11 55.8 ± 3.9 1539.9 ± 283.3 987.5 ± 74.6
Nec-1 273 ± 20 91.3 ± 6.3 1898.2 ± 359.3 1771.0 ± 170.1 249 ± 24 78.0 ± 6.9* 2200.7 ± 175.4* 1436.7 ± 138.4*
Nec-1i 279 ± 14 100.2 ± 10.0 2137.7 ± 171.9 1649.9 ± 172.4 251 ± 35 94.1 ± 10.7* 2504.7 ± 245.5* 1728.8 ± 155.4*
Nec-1s 257 ± 17 96.6 ± 9.0 2071.8 ± 290.6 1521.5 ± 138.2 282 ± 25 82.6 ± 7.7* 1807.8 ± 324.8 1335.0 ± 137.2*
GSK’772 262 ± 7 87.9 ± 4.4 2195.6 ± 188.0 1521.5 ± 138.2 282 ± 25 72.5 ± 5.0 1963.4 ± 235.5 1421.5 ± 178.8*
Data are presented as mean ± SEM; n = 6–8 per group
HR heart rate, LVDP left ventricular developed pressure, + /−(dP/dt)max maximal rates of pressure development/fall
*P < 0.05 vs. control group

inhibitors caused no such elevation in pSer282-cMyBPc/ cMyBPc ratio (Fig. 2a, g, m).
Assessment of oxidative stress

None of the investigated RIP1 inhibitors significantly affected the extent of lipid peroxidation in comparison to the control group. Nec-1 mildly increased the TBARS lev- els, while the effects of Nec-1i on this marker of OS were similar to those observed in controls (Fig. 3a). GSK’772 had a tendency to increase and Nec-1s decrease TBARS levels in comparison to the vehicle treatment (Fig. 3d). The extent of protein carbonylation was unchanged when comparing necrostatin-treated (Nec-1, Nec-1i, and Nec-1s) hearts with the vehicle and GSK’772 treatment tended to diminish OS in this regard. (Fig. 3b, e, g). Significantly elevated protein tyrosine nitration was found as a result of treatment with each investigated necrostatin (Fig. 3c, f, h). These adverse effects were not observed upon GSK’772 treatment (Fig. 3f, h).

Discussion
This study was conducted to assess the molecular effects of Nec-1 on heart function under non-cell death-related condi- tions and to find out whether other novel RIP1 inhibitors may also possess similar pharmacodynamics action. We indicated for the first time that Nec-1 is able to modulate the pThr287-CaMKIIδ/CaMKIIδ and pSer282-cMyBPc/ cMyBPc ratio, thereby potentially underlying its capabil- ity to enhance cardiac performance as documented by sig- nificant increase in contractile parameters LVDP and +(dP/ dt)max. Likewise, both intraperitoneal and intracoronary application of Nec-1 temporarily increased BP and short- ened the duration of the PR interval indicating faster con- duction of the impulses through the atria to the ventricles, thereby supporting its direct effects on ECC [11, 16]. Novel

RIP1 inhibitors (Nec-1s and GSK’772) seem to lack some of these effects. The former novel agent, Nec-1s, did not affect the tested Ca2+-associated molecular mechanisms while it also increased LVDP. The latter one, GSK’772, promoted the activation of CaMKIIδ-PLN axis with resultant positive lusitropic effects. In addition, GSK’772 was neutral with respect to the modulation of OS, while all necrostatins (Nec- 1, Nec-1i and Nec-1s) increased protein tyrosine nitration.
PKA and CaMKIIδ are key proteins regulating ECC by phosphorylation of their substrates and an interlink between the molecular signaling mediated by both these protein kinases is very complex [31]. Western blot analysis revealed that neither Nec-1 nor other RIP1 inhibitors used in this study affected the phosphorylation targets of PKA. Since this protein kinase plays a pivotal role in mediating the effects of β-adrenergic stimulation, it is unlikely that Nec-1 alters the sympathetic nervous system in the heart, and thus, changes in LV pressure and BP [11, 16] seem to be facilitated by other mechanisms. In contrast to PKA, Nec-1 treatment increased relative phosphorylation, thus activation of CaMKIIδ. The implication of CaMKIIδ in the regula- tion of the vascular tone, besides the heart, has been well documented using both in vitro [32, 33] and in vivo studies [34, 35]. Inhibition of CaMKIIδ either pharmacologically
[33] or genetically [32, 34, 35] has been shown to be associ- ated with lower BP in angiotensin II-induced hypertension. Given that, it is reasonable to hypothesize that the adverse effect of Nec-1 on BP might be mediated by CaMKIIδ via direct modulation of the vascular tone. Nonetheless, affect- ing the myocardium cannot be ruled out either, as this study demonstrated the changes in hemodynamic characteristics in Nec-1-treated hearts. In support, Nec-1 increased the CaMKII-mediated phosphorylation of cMyBPc at Ser282. Phosphorylated cMyBPc is of substantial importance for physiologic function of the heart and determines proper cardiac contraction via sarcomere mechanisms [36]. In con- trast, decreased levels of p-cMyBPc have been demonstrated in patients with challenging cardiac morbidities, like heart

Fig. 2 Analysis of expression of selected proteins of contraction– relaxation cycle in left ventricles lysates. Upper panel: RIP1 inhibi- tors prototypes; Lower panel: Novel RIP1 inhibitors; a Representative immunoblots and total protein staining. b–m Immunoblot quantifica-

failure [37] and atrial fibrillation [38]. Of note, besides Nec- 1, GSK’772 also increased the phosphorylation of CaMKIIδ. Intriguingly, however, pThr287-PLN as a downstream protein of CaMKIIδ was differently affected by these two agents. Nec-1 decreased, while GSK’772 increased the phosphoryl- ation of PLN at Thr17. Such different effects could indicate that due to Nec-1 treatment, the inhibitory effect of non- phosphorylated PLN on SERCA2a was preserved, eventu- ally leading to higher intracellular [Ca2+] and thus delayed relaxation of the heart. On the other hand, GSK’772 could directly promote lusitropy. In fact, the detailed analysis of timecourse of hemodynamic parameters has revealed that although both drugs increased −(dP/dt)max values at the 40th min, this effect was achieved earlier in GSK’772-perfused hearts.
OS has been known for many years as a conspicuous underlying mechanism of various heart defects [19, 39, 40]. Imbalance between excessive reactive oxygen and nitrogen species and antioxidant defense systems has been reported to cause myocardial damage through multiple mechanisms [17,

tion of PKA substrate (b, h), pSer16-PLN/PLN ratio (c, i), SERCA2a (d, j), pThr285-CaMKIIδ/CaMKIIδ ratio (e; k), pThr17-PLN/PLN ratio (f; l), and pSer282-cMyBPc/cMyBPc ratio (g; m). Data are pre- sented relatively to control group as mean ± SEM; *P < 0.05

19, 40]. In this study, we assessed OS by measurement of lipid peroxidation, protein carbonylation, and protein tyros- ine nitration. The extent of lipid peroxidation, end products of which are cytotoxic and can lead to cell signaling altera- tions and/or protein and DNA damage [41, 42], and protein carbonylation, which can irreversibly modify the expression and activity of proteins [43, 44], were unaffected by either RIP1 inhibitor in comparison to vehicle. On the other hand, each member of the necrostatins class elevated protein tyros- ine nitration, an adverse modification of proteins negatively affecting their function, turnover, and accessibility to other post-translational modifications, such as phosphorylation [43]. Since increased nitrosative stress is generally associ- ated with cardiovascular diseases via increased inducible nitric oxide synthase-mediated NO production [45], the observed adverse effect of necrostatins on protein tyrosine nitration may represent a serious off-target action of this drug class. In contrast, such disadvantageous effect on nitro- sative stress was not observed in GSK’772-treated group.

Fig. 3 Analysis of oxidative stress in left ventricles lysates. Upper panel: RIP1 inhibitors prototypes; Lower panel: Novel RIP1 inhibi- tors; a, d Analysis of membrane lipid peroxidation by means of TBARS assay. b, e Immunoblot quantification of protein carbon- ylation expressed as DNPH immunoblot positivity. c, f Immunob-

lot quantification of protein tyrosine nitration expressed as NO2-Tyr immunoblot positivity. g, h Representative immunoblots and total protein staining. Data are presented relatively to control group as mean ± SEM; *P < 0.05

In conclusion, this pilot study indicated for the first time that under non-cell death conditions, Nec-1 affects some proteins involved in the contraction-relaxation cycle and increases tyrosine nitration what may, at least in part, underlie its effects on LV pressure and BP. Broadly trans- lated, in addition to its direct effects on the myocardium, our findings can also indicate a possibility of Nec-1-mediated modulation of the vascular tone, but this hypothesis war- rants further research. Nec-1s, a more potent and stable necrostatin than Nec-1, comparably affected heart function, while it did not exhibit such effects on the selected proteins of Ca2+ cycling. Similar to Nec-1, it also elevated protein

tyrosine nitration. Importantly, we also partially highlighted the safety of GSK’772 by showing that this RIP1 inhibitor did not elevate nitrosative stress and augmented the activa- tion of CaMKII-PLN axis suggesting promotion of lusitropy. It is also important to note that the present study has not aimed to compare the selected RIP1 inhibitors in detail. The limitations of this study naturally include a defined profile of proteins regulating ECC and markers of OS. Likewise, the present evidence relies on acute treatment with the selected RIP1 inhibitors and does not assess effects of chronic admin- istration of these drugs. Nonetheless, these findings indicate that RIP1 inhibitors differently modulate heart function due

to various effects on redox signaling in cardiac cells and reg- ulation of some Ca2+-associated mechanisms. On one hand, the modulation of cardiac performance can be considered to be adverse effects, and on the other hand, it can maintain homeostasis of the cardiovascular system in certain patho- logical conditions. For instance, Nec-1-induced increase of BP can be considered as a side effect of this anti-necroptotic agent, while in conditions associated with hypotension, cir- culatory collapse, etc., such increase in LVDP could be ben- eficial. However, in this regard, it should also be mentioned that the potential use of this agent must be assessed with caution because it adversely modified the redox system. As GSK’772 elicited no such effects, this novel RIP1 inhibitor seems to be superior to Nec-1 as well as to other necrostatins increasing myocardial OS. Therefore, the latter agent could attract the most preclinical attention with a potential being further advanced for clinical examinations; however, further research is needed to explore the hypothesis we presented in this study.
Acknowledgements The authors would like to thank Ms. J. Formank- ova for her skillful technical assistance and Mgr. I. Jarabicova for her assistance with biochemical methods.

Funding This study was supported by The Slovak Research and Devel- opment Agency, Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic (APVV‐15‐607, APVV-20-0242, APVV-19-0540, VEGA SR 1/0016/20 and 2/0141/18).

Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval Protocol of this study has been approved by the Eth- ics Committee of the Faculty of Pharmacy, Comenius University in Bratislava. All procedures described herein were performed in accord- ance with the Guide for the care and Use of Laboratory Animals, pub- lished by the US National Institutes of Health (Guide, NRC 2011) and approved by the Animal Health and Welfare Division of the State Veterinary and Food Administration of the Slovak Republic.

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